Crystal Structure and Twisted Aggregates of Oxcarbazepine Form III

Polymorphism and crystal habit play vital roles in dictating the properties of crystalline materials. Here, the structure and properties of oxcarbazepine (OXCBZ) form III are reported along with the occurrence of twisted crystalline aggregates of this metastable polymorph. OXCBZ III can be produced by crystallization from the vapor phase and by recrystallization from solution. The crystallization process used to obtain OXCBZ III is found to affect the pitch, with the most prominent effect observed from the sublimation-grown OXCBZ III material where the pitch increases as the length of aggregates increases. Sublimation-grown OXCBZ III follows an unconventional mechanism of formation with condensed droplet formation and coalescence preceding nucleation and growth of aggregates. A crystal structure determination of OXCBZ III from powder X-ray diffraction methods, assisted by crystal structure prediction (CSP), reveals that OXCBZ III, similar to carbamazepine form II, contains void channels in its structure with the channels, aligned along the c crystallographic axis, oriented parallel to the twist axis of the aggregates. The likely role of structural misalignment at the lattice or nanoscale is explored by considering the role of molecular and closely related structural impurities informed by crystal structure prediction.

The temperature of evaporation for each experiment was controlled using a heating bath circulator by Huber (Peter Huber Kältemaschinenbau AG, Germany) which was filled with silicone oil. In experiments involving the addition of a substrate to investigate the effect on solid-form outcome, a 1 cm x 1 cm tile of commercial Al foil (Caterwrap®, exact purity and thickness unknown, Stephensons) was manually added to each crystallization vial after solution filtration had been carried out. The evaporation temperature, agitation-speed profile and sample holding time used in each experiment were programmed using the WinLissy software and all experiments were run using WinLissy's ZA Runner module. An overview of the experimental workflow implemented using the Crissy® platform is provided in Figure S1 and each experiment was typically performed in triplicate. A summary of all experiments and corresponding results are provided in Tables S1 and S2.

S3.1 Sublimation Experiments under Atmospheric Conditions
The sublimation experimental set-up is shown in Figure S4. The starting OXCBZ powder (form I) was sourced from Sigma-Aldrich UK (pharmaceutical secondary standard/certified reference material, purity 99.6%). Metallic substrates (1*1 cm) were attached to the glass slide and exposed to OXCBZ vapors as shown in in Figure S4.
The results from the sublimation are tabulated in Table S3.

S3.2 Sublimation under High Vacuum using QBox
Experimental outcomes from controlled sublimation studies conducted using a QBox 450 1 system (see section 2.2 in main manuscript for method details) are presented in Table S4. was employed. The correlation between pitch and length in one of the measured crystals is presented in Figure S7. Figure

S4.2 Twisted-Crystal Formation in OXCBZ Analogues
To investigate the possible emergence of twisted morphology for the structurally-

S5.1 Analysis of Samples from Sublimation Experiments
Representative XRPD patterns corresponding to a select number of samples derived from sublimation of OXCBZ, CBZ and CYT onto different experimental substrates are shown in Figure S11-S13. The sublimed materials were scraped onto low-background silicon sample holders and analyzed at room temperature using 1) a Bruker D8 Discover

S5.2 Analysis of Samples from Solution-Based Crystallization Experiments
Solution-grown samples were scraped onto low-background silicon sample holders and analyzed at room temperature using 1) a Bruker D8 Discover instrument operating at

S5.3 Variable Humidity XRPD (VH-XRPD) Analysis
VH-XRPD studies of OXCBZ III materials were conducted using the Bruker D8 Discover diffractometer, equipped with Anton Paar CHC plus⁺ Cryo and Humidity chamber and modular humidity generator MHG (ProUmid, Germany). Data were collected from 5 to 95% relative humidity (RH) in increments of 5% RH using a scan range of 3-40° 2θ, step size of 0.017° 2θ and count time of 1 s/step. All measurements were obtained at a temperature of 20°C and samples were held at each of the humidity profiles investigated for 15 minutes prior to data collection commencing.
The results obtained for the variable humidity study ( Figure S19) performed for the OXCBZ III material revealed no significant differences between the diffraction patterns collected at different relative humidity profiles, suggesting that form III remains stable and does not convert to the thermodynamically stable form I or the metastable form II when subjected to high-moisture uptake.

S5.4 Crystal-Structure Determination of OXCBZ Form III
The crystal structure of OXCBZ III was solved by real-space approach 7 using the simulated annealing algorithm as implemented within DASH. 8 (Table S5).

S8.1 Determination of Molar Absorption Coefficient (MAC)
A starting solution was obtained by dissolving OXCBZ form I sourced from Molekula in DMSO to achieve an initial solution concentration of 50 mM and latterly a 100 mM concentration. A Sirius Inform instrument (Sirius PAT2000i from Sirius Analytical, now Pion Inc., East Sussex) was utilized to obtain the MAC value by performing a pKa assay. 25 μL of solution were added to a vial and the Inform pKa assay was carried out using the Inform Control software as follows: 1) Initially, a blank reading was taken titrating from pH 2 to pH 12 in 40 mL of sodium chloride solution using hydrochloric acid and sodium hydroxide. 2) 33 mL of potassium chloride and 2 mL of acetate phosphate buffer were added in an automated fashion. 3) A total of 3 titrations were performed by the instrument going initially from low pH to high pH, then high pH to low pH and then ultimately low pH to high pH using hydrochloric acid and sodium hydroxide. Data were analyzed using the Inform Refine software, importing the blank aqueous reading to give a mean MAC value based on the molecular weight of OXCBZ which could be imported into the dissolution data set.

S8.2 Dissolution Measurements
Powder samples ( Data from GI dissolution studies comprising OXCBZ I and OXCBZ III compacts are presented in Figure S26. Across the pH range explored, the extrapolated dissolution rate was found to be higher for samples of form III when compared to pure form I material sourced from Molekula. Over the first ≈ 30 minutes of dissolution, the dissolution of compacts that included form III was found to be ≈ 2.6 times greater than those containing form I. The use of compacts with identical diameters in these studies ensured that differences in the particle size of forms I and III would not affect the dissolution outcome. OXCBZ III dissolving more readily than OXCBZ I is in line with past observations of metastable pharmaceutical polymorphs exhibiting improved dissolution compared to their more stable counterparts. 15 OXCBZ is a BCS Class II drug likely to exhibit dissolution rate-limited bioavailability 16 and, whilst the oral bioavailability profile of OXCBZ III was not investigated herein, it is anticipated that the superior dissolution of form III relative to form I will result in more favourable bioavailability properties and, potentially, more effective pharmaceutical formulations of OXCBZ. 15 Figure S26. pH dependent dissolution profiles of OXCBZ form I and form III.
Dissolution data were collected in the pH range of 2 -7.4 and the profiles depicted represent average measurements corresponding to 3 samples.

S9. High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS)
The OXCBZ starting powders undergoing sublimation for 16, 24, and 48 hours were additionally analyzed with HPLC-MS analysis. Prior to analysis commencing, blank samples were run to establish reference data for subsequent comparisons.
A representative chromatogram obtained from a blank run is shown in Figure S27a.
Comparison of the chromatogram of the OXCBZ reference material from Sigma-Aldrich UK with those of the powders used in sublimation experiments confirmed the presence of a chemical impurity (Table S6 and Figures S27b -S27e). The impurity had a retention time of ≈ 6.52 min in the liquid chromatograms with a corresponding m/z of 222.2 ( Figure S29) and was found to be present in all of the analyzed samples, including the reference material from Sigma-Aldrich. The impurity was subsequently identified as DBZ. The molecular structure of DBZ is shown in Figure S30.   (Table S6 and Figure S28). The analysis confirmed the presence of DBZ in both types of material, with the concentration of the impurity found to be less in the solution-grown material than in OXCBZ samples from sublimation experiments. Whilst the presence of DBZ in the solution-grown material was verified, its significance on the emergence of twisting for both the solution-and sublimationgrown crystals of OXCBZ III remained unclear.

S10. Conformation Analysis
The two conformers of the isolated OXCBZ molecule were optimized using  The anti conformer is found to be rather more stable than the syn conformer by 4.3 kJ/mol (PBE0/6-31G(d,p)), because of the position of the C=O group. The electrostatic potential around the conformers differs significantly with the anti conformer having a larger dipole moment ( Figure S31). The shapes of the two conformers are quite similar.

S11. Crystal Structure Prediction
Crystal structure prediction was carried out in the anti and syn conformation regions separately. As the anti conformer was seen in forms I and II, and is also more stable, one million (Z=1) initial structures in the 60 most common space groups were generated in the anti search region, and half a million in the syn region.   (Table S7 and Figure S33).
Form II was found 2.7 kJ/mol higher than form I (structure a20). Forms I and II are labelled with blue circles.
With the additional carbonyl group on the aromatic ring, the OXCBZ search found many structures with hydrogen-bonding motifs other than those usually seen in the carbamazepine family, i.e. amide C1,1(4) catemers or R2,2(8) dimers. OXCBZ form I was found amongst the lowest in lattice energy and the most densely packed structures. However, syn OXCBZ was able to form competitive structures despite being the less stable and less polar conformer ( Figure S33).

S12. Sensitivity of the Crystal Energy Landscape to Energy Models
The outcome of the CSP search is that OXCBZ can adopt a range of crystal structures, with two types of conformer, a variety of hydrogen bonding motifs and a significant variation in density, which are energetically competitive and within the plausible energy difference between polymorphs. Hence, we tested the sensitivity of the crystal energy models to the assumed energy model.

S12.1 Sensitivity to Polarization by the Environment
The sensitivity of the OXCBZ lattice energy landscape to the effect of the charge density changing on crystallization was estimated using a polarizable continuum model (PCM) with a representative dielectric constant (ε=3) for organic molecular crystals. 21 The CSP structures were re-optimized keeping the molecular conformations rigid with    24 and MBD* 25 schemes (Table S8).

S12.2 Periodic DFT-D Lattice Energies
The PBE-TS optimization improves the match between the structures a2 and a20 and the experimental structures, with a2 having an RMSD20 = 0.214 Å with form I (CANDUR01 determined at 95 K) and a20 a RMSD20 = 0.304 Å with form II (CANDUR02 also 95 K). However, the relative energies were very sensitive to the dispersion correction used, emphasizing the role of the van der Waals forces and the variation in density between the structures (Table S8).

S12.3 Sensitivity to Temperature
A simple estimate of the effect of temperature on the relative energies of the CSP   The overall comparison of the relative energies of the CSP crystal structures that match forms I (a2) and II (a20), the original global minimum, s59 and the structures that match the novel XRPD patterns (a96 and a165 for Form III) show significant variation with methods and the inclusion of temperature effects ( Figure S36). However, the variation is within the energy range of plausible polymorphism, though the new forms are calculated to be metastable.

S13.1 XRPD Patterns, Structures and Similarities
Five CSP structures, a96, a165, a722, a900 and a1858, were found to have XRPD patterns similar to those observed experimentally ( Figure S37and Table S9) and those reported by Lutker and Matzger. 26 Of these, a96 is likely to be within the energy range of plausible polymorphs, with a165 less stable, and the other three CSP structures considerably higher in energy ( Figure S35).
Among the five candidates, a96, a165 and a900 are in R-3 space group, with a165 loosely isomorphous to CBZ II, matching 15 out of a 15-molecule cluster, if the default similarity criteria of Mercury were lowered to 40% difference in distances and 40° in angles. a1858 is in P-3 group, while a722 is in hexagonal P61 group. However, these structures have similarities to each other matching 18 or more molecules within a 30molecule cluster, for a96, a900 and a1858, or between a165 and a1858 (Table S9).
There is less similarity between a722 and the other 4 candidate structures, and hence this structure was not discussed in the manuscript Figure 11.    Figure S38). Although a722 does not have the same channel construct as those in the trigonal structures, it has a similar construct of inter-channel packing. The diameters of void channels in a165, a722, a900

S13.2 Channel Properties
and a1858 are only slightly smaller than that of CBZ II which experimentally has been observed to contain molecules as large as toluene, 27 therefore these structures may be able to accommodate small solvent molecules which would stabilize the structures. The channels have a propeller-like directionality from the arched 2H-dibenzazepine rings: i.e. of the two OXCBZ phenyl rings, only those on the -NH2 side of the amide group line the hydrophobic interior walls of the channel. a96 a165 a722 a900 a1858 CBZ II CYT I Figure S38. Void channels in OXCBZ CSP candidates, compared to those in CBZ II, CYT I.

S13.3 Calculated Growth and BFDH Morphology of OXCBZ III Candidates
Geometry-based BFDH morphology of all OXCBZ III candidates, using CCDC Mercury, show that all will grow into hexagon-shaped needles, with similar dominant faces on the sides ( Figure S39). These BFDH calculated morphologies are in good agreement with the more elaborate growth morphology calculations for a96 and a165, which uses the forces between the molecules, here calculated from the ESP atomic charges and the FIT potential (i.e. the same intermolecular potential that was used for the CrystalPredictor search). The sides of channels are the slowest growing faces, while the ends channels have the fastest growth rate. In contrast, the growth and BFDH morphologies of form I and II of OXCBZ are more block-like ( Figure S40). Figure S39. BFDH morphologies of OXCBZ form III candidates a96, a165, a722, a900, a1858, also attachment-energy-based growth morphologies for a96 and a165.